间充质干细胞对多发性骨髓瘤细胞生长及硼替佐米诱导其凋亡的影响研究

Effect of mesenchymal stem cells on multiple myeloma cells growth and inhibition of bortezomib induced cell apoptosis

目的 探讨间充质干细胞在多发性骨髓瘤细胞生长以及硼替佐米诱导骨髓瘤细胞凋亡中的作用.方法 取15例临床确诊的多发性骨髓瘤（MM）患者以及3名正常供者的骨髓标本,分离其间充质干细胞（MM-BMSC和ND-BMSC）;测定细胞生长曲线、免疫表型和细胞因子分泌水平.将骨髓瘤细胞（NCI-H929）与BMSC细胞共培养,并加入蛋白酶体抑制剂硼替佐米,观察BMSC对NCI-H929细胞生长增殖的影响;进一步通过Annexin V-FITC/PI双染法流式细胞术（FCM）检测BMSC对硼替佐米诱导NCI-H929细胞凋亡的影响.结果 成功分离得到MM患者以及正常供者骨髓间充质干细胞,FCM检测显示两者均高表达CD73和CD105（＞95%）,表达CD44和CD29,不表达CD31、CD34、CD45和HLA-DR（＜1%）等表面分子.生长曲线测定显示MM-BMSC增殖较为缓慢,倍增时间（82 h）较ND-BMSC（62 h）略有延长（P＜0.05）.与ND-BMSC比较,MM-BMSC分泌高水平的细胞因子IL-6和VEGF,分别为（188.8±9.4）pg/ml对（115.0±15.1）pg/ml和（1497.2±39.7）pg/ml对（1329.0±21.1）pg/ml.将BMSC与骨髓瘤细胞NCI-H929共培养,MM-BMSC可促进骨髓瘤细胞的生存,降低NCI-H929细胞对硼替佐米的敏感性,明显抑制硼替佐米诱导的瘤细胞凋亡.结论 MM-BMSC可促进骨髓瘤细胞的生长,明显减少蛋白酶体抑制剂硼替佐米诱导的细胞凋亡.

Objective To investigate the role of mesenchymal stem cells（BMSCs）in multiple myeloma（MM）bone marrow（BM）microenrivonment and their effect on myeloma cells survival and bortezomib induced apoptosis.Methods BMSCs were derived from BM of untreated myeloma patients（MM-BMSCs）and healthy donors（HD-BMSCs）,respectively.The phenotype,proliferation time and cytokine secretion of MM-BMSCs were detected and compared with HD-BMSCs.Then BMSCs were co-cultured with myeloma cell line NCI-H929 and bortezomib in vitro.The NCI-H929 cells proliferation and bortezomib induced cell apoptosis were investigated.Results MM-BMSCs and HD-BMSCs were isolated successfully.The phenotype of MM-BMSCs was similar to that of HD-BMSCs.Expressions of CD73,CD105,CD44 and CD29 were positive,but those of CD31,CD34,CD45 and HLA-DR（〈 1%）negative.The proliferation time of MM-BMSCs was longer than that of HD-BMSCs（82 h vs 62 h,P 〈 0.05）.Moreover,over-expressions of IL-6 and VEGF in MM-BMSCs culture supernatant were detected as compared with that in HD-BMSCs [（188.8 ±9.4）pg/ml vs（115.0±15.1）pg/ml and（1497.2 ±39.7）pg/ml vs（1329.0±21.1）pg/ml,respectively].MM-BMSCs supported survival of the myeloma cells NCI-H929 and protected them from bortezomib induced cell apoptosis.Conclusions MM-BMSCs is benefit for myeloma cells proliferation and against cell apoptosis induced by bortezomib.Over-expression of IL-6 and VEGF maybe play a critical role in these effects.